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Study the role of LPS in induction of anti-tularemia immunity in humans and animals.Activity of various antigenic preparations of tularemia microbe, including highly purified from protein and S- and R-LPS, was studied using leukocytolysis reaction with blood of vaccinated humans and guinea pigs and skin allergy test (guinea pigs).Only the whole cells of Francisella tularensis, killed in protein non-denaturating conditions and conserving full S-LPS structure (tularin⁺) were shown to be inductors of delayed-type hypersensitivity reaction. Alterations in LPS structure (tularin⁻) results in a significant decrease, and denaturation of bacterial proteins (during boiling) results in a complete loss of immune stimulating properties of the preparations. Purified LPS preparations and O-polysaccharide fraction of S-LPS are not able to activate cell-mediated immunity.The presence of LPS with the full structure affects the ability of antigenic preparations of F. tularensis to cause allergic reactions, and thus, form cell-mediated antitularemia immunity. LPS of F. tularensis can not be excluded as an adjuvant and provides the most effective presentation of epitopes of protein molecules for interaction with receptors of T-lymphocytes.

Enhancement of tularemia laboratory diagnostics by F. tularensis DNA determination in blood sera of patients using real time polymerase chain reaction (RT-PCR).39 blood sera of patients obtained during transmissive epidemic outbreak of tularemia in Khanty-Mansiysk in 2013 were studied in agglutination reaction, passive hemagglutination, RT-PCR. Specific primers and fluorescent probes were used: ISFTu2F/R+ISFTu2P, Tu14GF/R+tul4-PR2.Advantages of using RT-PCR for early diagnostics of tularemia, when specific antibodies are not detected using traditional immunologic methods, were established. Use of a combination of primers and ISFTu2F/R+ISFTu2P probe allowed to detect F. tularensis DNA in 100% of sera, whereas Tul4G F/R+tul4-PR2 combination--92% of sera. The data were obtained when DNA was isolated from sera using "Proba Rapid" express method. Clinical-epidemiologic diagnosis oftularemia was confirmed by both immune-serologic and RT-PCR methods when sera were studied 3-4 weeks after the onset of the disease.RT-PCR with ISFTu2F/R primers and fluorescent probe ISFTu2P, having high sensitivity and specificity, allows to determine F. tularensis DNA in blood sera of patients at both the early stage and 3-4 weeks after the onset of the disease.

Detection of contemporary features of tularemia focimanifestations, determination of territories of high epidemic risk in various landscape zones and creation of a map of foci territories of Moscow Region for isolation of tularemia infectious agent cultures and registered human morbidity for justified planning of prophylaxis measures.Report materials of epizootologic examinations of natural foci for 1965-2013, 156 maps of epidemiologic examination of cases of human infection with tularemia, results of studies of casting of predatory birds and dung of predatory mammals were used. Registered morbidity and isolation of tularemia infectious agent cultures from 1965 to date were applied to an electronic map of Moscow Region by sign method using modern. GIS-technologies (MapInfo 10.5 program). Electronic maps Ingit at 1:200,000 scale, as well as Google Earth program were used to search for base points.Analysis of morbidity has revealed structure change in human tularemia morbidity--an increase of the fraction of urban population and a decrease of the fraction of patients among rural inhabitants, unimmunized against this infection are mostly ill. The presence of DNA of tularemia causative agent in biological objects in the complex with serologic and bacteriological studies was shown to allow to detect flaccid epizootics even at low numbers of rodents.Cartographic reflection of registered morbidity and isolation of tularemia infectious agent cultures allowed to show territories with various degrees of epizootic activity and epidemic manifestation. Positive results of serologic and molecular-genetic studies of environmental objects gives evident on epizootic activity and constant risk of aggravation of epidemic situation for this infection.

Comparative evaluation of the effect of exogenic serotonin on the development of apoptosis and proliferative activity of immune system cells of biomodels in vivo and in vitro in the dynamic of immunity forming against plague and tularemia.Analysis of relative content of immune competent cell DNA of unlinear and BALB/c mice was carried out after staining of the samples with mithramycin and ethidium bromide by flow cytometry.Administration of serotonin into biomodels before immunization with vaccine strains of Yersinia pestis and Francisella tularensis was established to increase in vivo proliferative activity of immune system cells, without a significant effect on their death by apoptosis. Serotonin inhibited in vitro the development of apoptosis of mice blood leukocytes in response to both the vaccine Y. pestis EV strain and tularin.Biogenic amine serotonin shows equivalent modulating effect on both anti-plague and anti-tularemia immune response in vivo and in vitro, without disrupting immune system homeostasis.

Comparative analysis of parameters of humoral and cell immunity in individuals, vaccinated against tularemia.Sera and blood samples of 258 immunized individuals were studied by indirect hemagglutination and leukocytolis with tularin reaction.73% of the examined individuals had both specific antibodies and positive values of cell immunity. The presence of anti-tularemia immunity was registered in 26 of 30 individuals immunized 10 - 20 years ago. However in 76 individuals (26%) we have detected discrepancies of the results of the 2 methods that complicate the evaluation of specific immunity status. As such, the use of only 1 method characterizing either humoral or cell immunity does not give objective information.The use of 2 methods directed on detection of both specific antibodies and delayed-type hypersensitivity reaction is reasonable for the increase of validity of results of anti-tularemia immunity status evaluation. Only positive immunologic parameters of both tests confirm the presence of immunity against Francisella tularensjs and the possibility of revaccination period delay.

Determination of penicillin resistance features in tularemia causative agents of the mediasiatica subspecies, stability evaluation of differences in strains of various taxa and development of a rapid method of F. tularensis intraspecies differentiation.Beta-lactamase activity was determined in 30 strains of Francisella genus bacteria by quantitative iodometric method.All the strains regardless of subspecies membership were characterized by high resistance to beta-lactam antibiotics, and bacteria of the mediasiatica subspecies in contrast to other francisella did not synthesize beta-lactamase. Our attempts to induce beta-lactamase activity in vitro and in vivo in strains of this subspecies did not succeed. A method of intraspecies differentiation of F. tularensis by nitorcefin disks is proposed based on the distinctive feature.A high level of F. tularensis subsp. mediasiatica resistance to beta-lactams in vitro and their inefficiency during therapy of experimental tularemia due to a beta-lactamase negative strain suggests that F. tularensis beta-lactamase is not the leading factor in formation of native penicillin resistance of tularemia causative agent.

Morphometric characteristic of organ and system state of guinea pigs immunized with live tularemia vaccine during infection with virulent culture of tularemia.Morphometric study of histological material from immunized guinea pigs infected subcutaneously at day 30 with a culture of virulent tularemia strain was performed. A standard scheme of sampling and preparation of morphological material and staining of final semifine section with hematoxylin and eosin, impregnation with silver by Masson in Gamperl and Grimelius modificationwas used. Morphometric study was performed by using "Densitomorphometry" program.Morphometric parameters that characterize functional state of organs and systems in immunized, immunized with consequent infection and infected guinea pigs were established. Reactive processes that take place in the infected animal organism against the background of prior immunization fit into the range of adaptation-compensation reactions.The morphometric study carried out allowed to adequately evaluate the state of functionally important systems of the organism of experimental animals, this allows to consider perspective the wider use of morphometric analysis in experimental morphology.

Study of the current spread of natural tularemia foci in Mongolia and its epizootic activity evaluation for consequent substantiation of the recommendations for prophylaxis of this disease.Study of 1119 pellet specimens from predatory birds obtained in 6 aimag in Mongolia in 2008--2010 was performed. Tularemia antigen was detected by using antibody neutralization reaction (ANR) and passive hemagglutination reaction (PHR) with tularemia diagnosticums. Tularemia DNA was detected by PCR by using strain specific primers. Presence of plague antigen in PHR with plague immunoglobulin diagnosticum was also studied in all the samples.Epizootologic monitoring allowed the detection of natural tularemia foci in 5 of the 6 studied aimags in Mongolia. PHR was the most effective study method that allowed to detect tularemia antigen in the environmental objects in high quantities (up to 9.2% of positive samples) and high titers (up to 1:1600). PCR was less effective. Plague antigen was detected in 9 samples in 2010 for the first time, and in 3 cases together with tularemia antigen, which indicates a presence of combined natural foci of tularemia and plague in this territory.In the studied regions of Mongolia natural tularemia foci were detected, their epizootic activity was determined and recommendations for future study tactics of natural tularemia foci were given.

Results of virological and bacteriological studies of wild mammals of 11 species from Rodentia and Cricetidae genuses during epizootic period (spring-autumn 2006-2007) in Murmansk region are presented. The number of red-baked mice (Clethrionomys) and common vole (Microtus) was rising. Antigen of hemorrhagic fever with renal syndrome virus as well as tularemia pathogen were found in background rodent species.

For the first time, temperate Legionella bacteriophage was isolated from organs of guinea pig infected with Philadelphia 1 strain of Legionella pneumophila. Negative colonies of bactriophage from 1.5 to 2.5 mm in diameter were detected. Central part of them was transparent and surrounded by peripheral zone of partial lysis. Electron microscopy showed that corpuscles of the phage consist from multifaceted elongated head of stretched hexagonal form and short tail. The bacteriophage lyzed bacteria, which cause Legionnaires' disease, and also had certain lytic activity against causative agent of tularemia.

Investigation of ability of Francisella tularensis S- and R-lypopolysaccharide (LPS) preparations as well as the live bacteria with different chemotypes to interact with human lypopolysaccharide-binding protein (LBP) was carried out. It was found that LPS preparations derived from virulent(S-LPS) or isogenic avirulent mutant (R-LPS) strains of F. tularensis had markedly lower affinity to LBP as compared with typical S-LPS of Salmonella abortus and R-LPS of Yersinia pestis. It was shown that R-LPS preparation from avirulent mutant binds LPB more effectively than S-LPS from F. tularensis virulent strain. Differences in S- and R-LPS affinity were also confirmed for LPS represented by the live cells. Thus, bacteria with S-chemotype of LPS (F. tularensis 15/10) bound only 20.3% of LBP, whereas cells with R-LPS (F. tularensis 543 cap(-)) bound 39.9%. Such pattern was observed in experiments with both normal non-immune human serum and sera from people immunized with live tularemia vaccine. The latter indicates that opsonization of LPS by specific antibodies does not change its affinity to LBP. The observed more efficient binding of avirulent strain R-LPS to LBP is likely determines the more intensive host response directed to destruction and rapid elimination of the causative agent. At the same time, weak affinity of the vaccine and virulent strains S-LPS to LBP probably allows the bacterium to avoid activation of host defense mechanisms thus contributing to its long-term persistence in microorganism and development of specific immunity against tularemia.

Data on influence of Francisella tularensis C-complex preparations on formation of immunity against tularemia are presented. Study of cellular immunity characteristics as well as dynamics of antibody response was carried out on white mice and guinea pigs models. Absence of toxicity, pyrogenicity, and negative effects on immunocompetent cells in combination with protective activity points to possibility of use the C-complex as a component of a subunit vaccine.

A highly purified TUL4-CBD chimeric protein was obtained by one stage purification method. TUL4-CBD protein consists of TUL4 Francisella tularensis mature peptide sequence, Gly-Ser spacer and cellulose binding domain (CBD) of Anaerocellum thermophilum. The TUL4-CBD protein was shown to induce production of specific antibodies to TUL4 protein in laboratory animals.

Retrospective VNTR-analysis of 159 Francisella tularensis subsp. holarctica strains isolated in December 1988 - February 1989 in former USSR and some European countries was carried out. Analysis of heterogenic genotypes of strains allow to subdivide them into 30 groups of variants by individual genotypes, while cluster analysis--to subdivide them in 7 clusters with different number of compositions. The predominance of genotype C1 strains isolated on the Rostov and Archangelsk regions and the Crimea was established. F. tularensis strains isolated in winter time 1988 - 1989 in different geographic regions were supposed to be resident cultures typical for their biotope in natural focus of disease.

The study of the persistence potential of 64 F. tularensis strains isolated from different sources was carried out. The wide spread of the antilysozyme, antilactoferrin and anticomplementory activities of F. tularensis were detected. F. tularensis, isolated from ticks and water, were characterized by the highest level of the expression of antilysozyme activity, while anticomplementory and antilactoferrin activities of the infective agents were characteristic of those microorganisms which were isolated from rodents and their excrements.

This work deals with the results of 40-year observations on the circulation of infective agents in the natural foci of infections (tularemia, leptospirosis, hemorrhagic fever with renal syndrome) among rodents and other small mammals in the territory of the Moscow. The monitoring of their frequency and the infection rates remains the main effective measure for the prophylaxis of dangerous infections among the population of the megapolis.

The comparative study of the specificity of antibodies in human sera after tularemia infection and immunization with live tularemia infection was carried out with the use of passive hemagglutination and immunoblotting techniques. The sera of tularemia patients contained two different types of immunoglobulins: strictly specific to the antigenic epitopes of F. tularensis Iipopolysaccharide (LPS) and strictly specific to F. tularensis subsp. novicida LPS. Such phenomenon may be due to phase variations of the antigenic structure of F. tularensis LPS in the body of a slightly susceptible host. The immune sera of vaccinated were found to contain antibodies, strictly specific only to F. tularensis LPS. At the same time in one vaccinee by the presence of pronounced postvaccinal reactions was found sharply defined interaction between serum imunoglobulins and F. tularensis subsp. novicida LPS. As the result, the data on the possibility of the antigenic modification of F. tularensis in tularemia infection in humans were obtained. At the same time antigenic epitopes, characteristic of faintly pathogenic and closely related F. tularensis novicida LPS, appeared in the structure of F. tularensis LPS.

Cross-reacting antigens in B. mallei, B. pseudomallei, B. thailandensis, Francisella tularensis, Yersinia pestis and Mycobacterium tuberculosis were studied with the use of immuno- and electrophoretic techniques. The set of antigens was shown to be almost identical in the causative agents of glanders, melioidosis, as well as in B. thailandensis, though in the latter organism 200-kD glycoprotein was absent. The analysis of immuno- and proteinograms demonstrated the presence of cross-reactions in the representatives of the genus Burkholderia with the causative agents of plague, tularemia and tuberculosis, which served as the basis for making the scheme of their antigenic relationships. The use of immunosorption techniques with subsequent analysis of the preparations by means of the SDS polyacryl gel electrophoresis and immunoblotting made it possible to characterize cross-reacting antigens of the pathogenic microorganisms under study, to establish their molecular weights (81-15 kD) and to show that some detected antigens are analogous to B. pseudomallei outer membrane proteins (34 and 30 kD).

In experiments on guinea pigs immunized with Francisella tularensis 15, or live tularemia vaccine (LTV), the level of heterologous protective effect to dangerous infectious diseases caused by Yersinia pestis, Burkholderia pseudomallei, B. mallei, Mycobacterium tuberculosis was studied. The study revealed that during the first 4 weeks after the subcutaneous immunization with LTV the level of resistance of the immunized animals to heterologous infective agent reliably increased as indicated by the survival rate of the animals, as well as by the survival time of those killed by infection, in comparison with the controls. Later (on day 150 after immunization) differences in death rate between the groups perceptibly decreased. Nevertheless, the 1 1/2-fold increase of the survival time of the challenged immunized animals in comparison with the controls proved the possibility of using immunization with LTV for the urgent prophylaxis and treatment not only of tularemia, but also of plague, glanders, melioidosis and tuberculosis.

In the analysis of F. tularensis genome with the use of the specially developed program "DNA" a great number of loci containing tandem repeats were found. For analysis, 3 of them were selected and designated as FtA, FtB, FtC. The study of DNA of 40 F. tularensis strains in the polymerase chain reaction with specific primers to these loci a great variability in the number of repeats was established, the presence of 17 alleles being found in locus FtA, 5 alleles in locus FtB and 5 alleles in locus FtC. The strains under study formed 24 variants of genotypes, whose occurrence varied from 0.025 to 0.125. Taking into account the variability of the detected loci and a great number of potential loci VNTR in the genome, further development of this method will facilitate the creation of local and general data bases of the strains, thus ensuring more effective genetic typing of F. tularensis.

The matrix for obtaining silica-based diagnosticum was selected, its activation with surfactant was optimized and its immobilization with tularemia immunoglobulins was carried out. In the glass suspension agglutination test (SAT) the sensitivity of the diagnosticum was 3.125 x 10(6) to 6.25 x 10(6) microbial cells per ml; the duration of SAT, including the evaluation of its results, was 1-5 minutes.

The data on the epidemiological and epizootological manifestations of tularemia on the territory of the Ulyanovsk region are presented. The characteristics describing the process of the of Francisella tularensis circulation as well as the environmental objects, most important for the manifestations of tularemia infection, are given.

Under conditions of artificial water biocenosis a virulent strain of F. tularensis could be detected in fresh water shrimps and mollusks for about a month, in Conepoda for up to 20 days and in Chydorus sphaericus for up to 7 days from the moment of the aquaria water contamination. In silt F. tularensis could be detected for a longer period (up to 2 months). Daphnia, Oligochaeta and C. sphaericus appeared to be unfavorable environment for this microorganism. The virulence level of F. tularensis microbial cells decreased in paralell with prolongation of their stay in water biocenosis. The presence of water biota favours F. tularensis preservation in water reservoirs for a longer time.

To determine antitularemia antibodies in the sera of humans and animale, the possibility of using dot immunoassay with the use of F. tularensis lipopolysaccharide (LPS) as antigen-containing preparation was ascertained. Experiments demonstrated that this method made it possible to determine specific antitularemia antibodies in the sera of sick and immunized humans and animals. Investigetions carried out with the use of heterologous antisera to F. novicida, F. novicida-like and F. philomiragia, as well as Brucellf abortus, Vibrio cholerae and Yersinia enterocolitica, revealed that F. tularensis S-LPS was highly specific. The results obtained in this investigation are indicative of good prospects of using F. tularensis LPS in dot blotting for the laboratory diagnostics of tularemia in humans.

The electron-microscopic study of the interaction of F. tularensis virulent and attenuated strains with infusoria of the species T. pyriformis was dynamically studied. In this study the structural changes of F. tularensis and T. pyriformis cells, as well as their capacity for survival, were revealed. The data on their ultrastructure correlated with the dynamics of the number of both F. tularensis and T. pyriformis: during the whole term of observation the tendency to a slow decrease in the number of F. tularensis was registered with the concentration of T. pyriformis remaining stable. The interaction of F. tularensis with T. pyriformis may be regarded as a variant of commensal, but not antagonistic interactions.

The effectiveness of immunization with Burkholderia pseudomallei attenuated strains (Pur and Ts), heterologous vaccines and the recombinant culture of Francisella tularensis RM2 carrying a plasmid with fragments of B. pseudomallei chromosome was studied in four species of experimental animals, essentially differing in their sensitivity to melioidosis. The most immunogenic B. pseudomallei mutants, introduced subcutaneously, created a statistically significant level of protection in animals, moderately sensitive to melioidosis, but proved to be ineffective in highly sensitive animal models when tested under the same conditions. In aerogenic infection the effectiveness of the experimental vaccines under study in all species of the animals was on the same level. The study showed good prospects of using tularemia vaccine for inducing heterologous immunity to melioidosis, as well as the possibility of its use as the basis of a bivalent gene-engineering vaccine.

Conditions for the appearance of F. tularensis uncultivated forms and for their reversion into the initial state have been studied. As revealed in this study, the combined influence of stress factors (starvation and low temperature) may result in the transition of F. tularensis into the uncultivated state in which it persists in the environment during the period between epidemics. The reversion of F. tularensis uncultivated forms into the initial state has been carried out with the use of sensitive animals. The uncultivated state of F. tularensis should be regarded as the actual form of the existence of the causative agent of tularemia in soil and water ecosystems.

The effect of immunization with Burkholderia pseudomallei, (Pur- and Ts), heterologous vaccines and the recombinant culture of Francisella tularensis RM2, carrying a plasmid with fragments of B. pseudomallei chromosome, was studied on four species of experimental animals, essentially differing by their sensitivity to melioidosis. B. pseudomallei mutants formed the statistically significant level of protection in subcutaneously challenged animals, moderately sensitive to melioidosis, but were not effective when tested, under the same conditions, in animals, highly sensitive to melioidosis. The effect produced by the experimental vaccines under study in animals of all species, subjected to aerogenic challenge, was leveled. The study showed good prospects for the use of tularemia vaccine with a view to create heterologous immunity to melioidosis and the possibility of its use as the basis of bivalent gene engineering vaccine.

For the first time F.tularensis recombinant strain R1A, obtained by the transfer of genes responsible for virulence in F.tularensis strain B 399 A-Cole into recipient cells of the R-form, was studied. Strain R1A was characterized by morphological, tinctorial and biochemical properties, similar to those of F.tularensis virulent strains, and became resistant to the bactericidal action of animal sera, as well as heat resistant (tr42). The results of animal experiments revealed that strain R1A proved to be highly virulent for noninbred white mice, faintly virulent for guinea pigs and avirulent for rabbits. In contrast to the R-form, recombinant strain R1A exhibited high antigenic activity, as well as partially protected guinea pigs from 100 DCL of F.tularensis highly virulent strain B A-Cole. The totality of its properties places recombinant strain R1A in an intermediate position between F.tularensis virulent and vaccine strains.

A rapid and simple method for the in vitro evaluation of the virulence of F. tularensis is proposed. The optimal parameters--the concentrations of bacteria and the substrate (phenolphthalein phosphate), the time incubation in the serum--have been developed and special conditions for strains of different subspecies have been selected. The proposed method makes it possible to greatly reduce the time of obtaining results and to exclude labor-consuming experiments on laboratory animals when working with large collections of F. tularensis laboratory and natural wild strains. Moreover, the method widens the possibilities of experimenters in the evaluation of the effect of some mutation on the virulence of the strain under study.

Cases of tularemia were registered in the Crimea both before and after planned immunization. In 1981-1993 in 4.000 localities 35,100 mammals, 27,400 ectoparasites, 8,800 feces left by birds of prey and foxes and 900 environmental specimens were studied. 137 Francisella tularensis strains were isolated. Field mapping of the spread of F.tularensis and places of habitation of small mammals was carried out. The active polyhostal natural focus of tularemia was found to exist on the Kerch Peninsula, less affected by anthropogenic factors, where tularemia morbidity among humans, and tularemia epizootic coincided with the maximum rises in the number of myomorphs. The core of the focus constituted 2.4% of its area and were characterized by the stable complex of Ixodes ticks and the preservation of F. tularensis in the environment. In other regions of the flat part of the Crimea with considerable anthropogenic transformations of the landscape rises in tularemia epizootics and tularemia morbidity in humans were rare. In the mountainous part of the Crimea tularemia epizootics were registered only by the detection of specific antibodies and antigens.

Monoclonal antibodies to F.tularensis cells, subspecies holarctica, were studied for the capacity of reacting with F.tularensis, subspecies nearctica, and its mutants having lower virulence and altered capacity for inducing protective immunity to tularemia in laboratory animals. Among the antibody-producing hybridoma clones under study, clones F8/67 and C7/65 capable of distinguishing the mutants of F.tularensis, subspecies nearctica, with lower virulence than that of the initial strain were selected. Antibodies of these hybridoma clones did not interact with the antigens of the initial virulent strains of F.tularensis, subspecies nearctica, while giving pronounced reaction with the antigens of its mutants. Close F8/67 produced IgG antibodies and clone C7/65, IgM antibodies. As shown in immunoblotting, antibodies produced by these hybridoma clones bound with proteins of F.tularensis cell membranes.

The natural isolate of F. tularensis subsp. holarctica 268 was detected and studied. The isolate possessed the properties of the vaccine strain: residual virulence for white mice, avirulence for guinea pigs and high immunogenicity for experimental animals. A significant decrease in its virulence for animals, highly sensitive to tularemia, was noted. In contrast to most virulent strains circulating in natural foci, F. tularensis strain 268 was characterized by the absence of growth at a cultivation temperature of 42 degrees C and had stable biological properties after 10-fold passage through white mice.

In this investigation isogenic avirulent variants obtained from F. tularensis standard virulent strain 503 were used. The capsule-deficient variants (cap-) were shown to have no species-specific capsular antigens and to be capable of producing R-LPS having no the polysaccharide part of the molecules. The capsule-defective forms (cap +/- ) were found to synthesize capsular antigens and S-LPS whose polysaccharide part essentially differed from the O-lateral chains of LPS of the virulent strain. The study of bacterial dissemination revealed that virulent bacteria rapidly spread in the macroorganism, and their subsequent proliferation shortly led to the death of animals. Avirulent mutants (cap- and cap +/- ) appeared in the organs of animals later and proliferated slower, parasitizing in the macroorganism without fatal outcome. The cap- variants were not capable of inducing the synthesis of antitularemic antibodies and possessed no protective properties. The cap +/- mutants were capable of inducing the synthesis of antitularemic antibodies in mice. These antibodies facilitated the elimination of avirulent strains from the macroorganism, but did not ensure protection from infection with virulent strains.

Primers for the detection of F.tularensis in the polymerase chain reaction (PCR) have been obtained. The method of PCR permits the identification of up to 10 F.tularensis cells in the analysis of pure and mixed cultures, as well as suspensions prepared from the organs of laboratory animals. The possibility of the simultaneous analysis of several samples makes this system for the detection of F.tularensis useful for epidemiological investigations.

The comparative study of newly discovered pathogenic bacteria of the genus Francisella was carried out with the use of a complex of microbiological and serological methods. While having great similarity to the causative agent of tularemia, F. novicida, F. novicida-like bacteria and F. philomiragia had lesser growth requirements, some specific morphological and structural features, were capable of fermenting sucrose and exhibited low pathogenicity to experimental animals. The strains under study proved to be virulent with regard to golden hamsters, who were for this reason proposed as an adequate model for the isolation of these bacteria from environmental objects and pathological material obtained from patients. The use of immunoblotting made it possible to find out that all Francisella species had protein antigens, similar to their electrophoretic mobility and serological activity.

The influence of exogenic human recombinant tumor necrosis factor (TNF-alpha) on antibody production in mice immunized with the preparation of F.tularensis outer membranes (OM) was studied. TNF-alpha was injected into mice in doses of 0.001-10 I.U. before and simultaneously with the injection of the preparation of F.tularensis OM. The levels of tularemia antibodies, determined by ELISA techniques, and the number of antibody-producing cells (APC) were studied. The study revealed that recombinant TNF-alpha in the range of doses used in this investigation stimulated the formation of humoral immunity. The injection of TNF-alpha in a dose of 0.001 I.U. was found to produce the most pronounced effect on the level of tularemia antibodies and the number of antibody-producing cells. The use TNF-alpha as immunomodulator made it possible to decrease the dose of the preparation of F.tularensis OM introduced as immunogenic agent without essential changes in the number of splenic APC and in the level of tularemia antibodies.

The activities of NADP-oxidase and glucose-6-phosphate dehydrogenase in mononuclear phagocytes and polymorphonuclear leukocytes (PML) during the phagocytosis of Francisella tularensis and the influence of immunization of on the activity of oxygen-dependent metabolism were studied. The phagocytosis of F.tularensis was found to be accompanied by a rise in the activity of NADP.H-oxidase and glucose-6-phosphate dehydrogenase. In the process of immunogenesis the rearrangement of the enzymatic system ensuring the antimicrobial action of phagocytes occurred. Macrophages exhibited higher enzymatic activity than PML.

In this work the conditions of labeling protein A with europium ions were studied and the conjugates obtained in this study were compared with traditional peroxidase conjugates currently used in immunochemistry. The conjugates of protein A with Eu3+ chelate were obtained with the use of cyclic dianhydride of diethylenetriaminepentaacetic acid (DADETPA). Conjugation methods with the use of DADETPA was shown to permit obtaining high-quality conjugates with europium chelates. Europium-labeled protein A ensured the sensitivity of the determination of adsorbed IgG at a level of 2 ng/ml and the dynamic analytical range within 3-1,000 ng/ml, which essentially exceeded similar characteristics of peroxidase conjugates with protein A. Europium-labeled protein A was used for the detection of antibodies to Francisella tularensis in the sera of humans immunized against tularemia. The sensitivity of this assay exceeded that of the enzyme immunoassay 10- to 40-fold. A conclusion was made on the possibility of using europium labelled protein A for the determination of specific antibodies to F.tularensis. This preparation may be useful in the determination of specific antibodies in low-immune sera.

The protective properties of the preparation of F. tularensis outer membranes (OM), obtained from F. tularensis vaccine strain 15, were studied in experiments on hamadryas baboons challenged subcutaneously with F. tularensis virulent strain Schu (nonarctic subspecies). The subcutaneous immunization with the OM preparation prevented the development of clinically pronounced infection in more than 70% of the monkeys challenged with F. tularensis strain Schu in a dose of 787 live microbial cells 30 days after immunization. Antibody titers determined in the immunized monkeys with the use of the agglutination test (AT) and the passive hemagglutination test (PHAT) were usual in minimal diagnostic limits (1:80 for AT and 1:320 for PHAT) and did not significantly rise by day 20 after immunization. In all intact animals infected with F. tularensis strain Schu the development of the infectious process was registered, which was accompanied by a rise in temperature exceeding 39.5 degrees C and a rise in the titer of specific antibodies.

In this study a search for F. tularensis antiphagocytic factor and an attempt for its isolation were made. For this purpose a fraction of F.tularensis water-saline extract saturated 55-60% with ammonium sulfate was split into separate components by preparative methods. One of them, consisting of two antigens of glycoprotein nature, had the capacity of decreasing the digestion of F.tularensis by guinea pig peritoneal macrophages about 10 times. The study revealed that the protein of the preparation obtained in this investigation was capable of suppressing digestion, thus facilitating the active proliferation of F.tularensis inside the cells of the host body.

The immunological evaluation of the influence of individual gel-chromatographic antigenic fractions (GAF) of F. tularensis outer membrane on different forms of T-cell reactiveness, such as delayed hypersensitivity (DH), proliferation of lymphocytes in the reaction of blast transformation (RBT) and mixed lymphocyte culture (MLC), has been made. As revealed in this study, GAF isolated from F. tularensis produce a pronounced immunomodulating effect on the processes linked with polyclonal activation of T-lymphocytes. Thus, GAF II with a molecular weight of 85-200 kD inhibits the maturation and activity of T-effectors of DH, the proliferation of lymphocytes in RBT and MLC. On the contrary, GAF IV with a molecular weight of 15-35 kD produces a stimulating effect on T-cells in the immune system in all the parameters under study.

Subcutaneous immunization, made in a single injection, with outer membrane preparations obtained from F.tularensis vaccine strain 15 and virulent strain A'Cole results in intensive immunity to tularemia in guinea pigs, ensuring the protection of 60-100% of the animals within a month after challenge with F.tularensis virulent strain 503 in a dose of 1,000 DCL. The development of protective effect induced by F.tularensis outer membranes can be observed during the first 24 hours and reaches its maximum by days 15-21 after immunization.

The influence of different gel-chromatographic antigenic fractions (GAF) of the membrane of F. tularensis, strain A'Cole, on different forms of reactivity of mouse peritoneal macrophages, such as the adhesion, ingestion and presentation of antigen on the cell surface, has been immunologically evaluated. GAF isolated from F. tularensis have been shown to produce a pronounced modulating effect on all forms of macrophagal functional activity under study. Thus, GAF II with a molecular weight of 85-200 kD inhibits the adhesion, ingestion and presentation of antigens and, on the contrary, GAF IV with a molecular weight of 15-35 kD stimulates these functions.

Comparative study of the properties of initial capsular (cap+) and non-capsular (cap-) F. tularensis strains has revealed that the non-capsular variants have their specific biological features. The characteristic features of these variants are avirulence and inability to produce an immunogenic effect in sensitive laboratory animals. Cap- mutants, depending on the method of their obtaining, may exhibit different sensitivity to some antibacterial preparations. In contrast to the initial virulent strains, cap- variants are intensively phagocytosed by peritoneal macrophages of white mice and capable of multiplication and survival in these macrophages. F. tularensis non-capsular forms are characterized by higher superoxide-dismutase and neuraminidase activity than the initial strains, which may be the explanation of the phenomenon of their intracellular survival. At the same time the main biochemical signs of cap- strains (citrullinureidase, phosphatase and penicillinidase) remain unchanged, which determines the possibility of their classification with this subspecies.